In previous work I found it possible to prepare and maintain cultures of dissociated embryonic chick or mouse dorsal root ganglion (DRG) neurons essentially free of nonneuronal cells. Extracellular electrophysiological techniques and radioautographic assays for DNA, RNA, and protein synthesis have been employed in initial characterization of these cultures. The research being undertaken on the project will attempt to extend the experimental utility of this neuronal cell culture system by exploring methods likely to provide cultures of spinal cord (SC) neurons analogous to the DRG cell cultures and by continuing development of analytical approaches to the characterization of DRG, SC, and mixed DRG/SC neuronal cultures. Use of H3-thymidine-labeled donor embryos and subsequent culture radioautography to define "birthdate spectra" for neurons surviving in culture, combination of extra- and intracellular electrophysiological techniques in a "screening test" for functional synaptic connections in vitro, and immunohistological staining to distinguish types of neurons in mixed cultures will be explored in efforts to answer such questions as whether the culture techniques select for particular sub-populations of neurons from the tissues origin, whether synapses between neurons can form in the total absence of nonneuronal cells, and whether the synapses formed, if any, exhibit the interneuronal specificities and physiological properties that obtain in vivo. The longer range objectives of the project involve efforts to define the synthetic activities necessary for synapse formation and maintenance and to initiate experimental approaches directed toward discovery of the molecular mechanisms underlying synaptic specificites and function.